Dimethylsulfoniopropionate-Dependent Demethylase (DmdA) from Pelagibacter ubique and Silicibacter pomeroyi

Abstract

The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria Pelagibacter ubique HTCC1062 and Silicibacter pomeroyi DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The P. ubique enzyme possessed an M r on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the M r was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both S. pomeroyi and P. ubique possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent K m s for DMSP were 13.2 ؎ 2.0 and 5.4 ؎ 2.3 mM for the P. ubique and S. pomeroyi enzymes, respectively. In cell extracts of S. pomeroyi DSS-3, the apparent K m for DMSP was 8.6 ؎ 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.

Structures of dimethylsulfoniopropionate‐dependent demethylase from the marine organism Pelagabacter ubique

“…As expected, it has been demonstrated that DmdA also has a requirement for the cofactor tetrahydrofolate (THF). 6,[18][19][20] However, in comparison to these previously characterized enzymes a significant mechanistic difference is the observation that 5-methyl-THF is the product of the DmdA reaction.…”

“…13 Initial insight into the function of DmdA comes from sequence searches that indicate that this enzyme is most closely related to the glycine cleavage system T-protein, dimethylglycine oxidase, and sarcosine oxidase. 6 Structures for most of the latter enzymes are available in the PDB, in both the apo form and with various ligands bound. [14][15][16][17] Although the residues of identity vary for each comparison, the sequence identity for the clycine cleavage system T-protein, dimethylglycine oxidase, and sarcosine oxidase is 26, 24, and 22%, respectively.…”

“…Dimethylsulfoniopropionate-dependent demethylase A (DmdA), the enzyme responsible for the demethylation of DMSP, has been recently isolated and characterized from two marine bacterial species Ruegeria pomeroyi and Pelagibacter ubique. 6 In subsequent steps, MMPA can be further demethylated or demethiolated to form either mercaptopropionate or methanethiol. 6,8,13 The rapid incorporation of methanethiol into the proteins of marine bacteria is well established.…”

“…6 In subsequent steps, MMPA can be further demethylated or demethiolated to form either mercaptopropionate or methanethiol. 6,8,13 The rapid incorporation of methanethiol into the proteins of marine bacteria is well established. 13 Initial insight into the function of DmdA comes from sequence searches that indicate that this enzyme is most closely related to the glycine cleavage system T-protein, dimethylglycine oxidase, and sarcosine oxidase.…”

“…5 Marine bacterioplankton are considered to be the primary mediators of DMSP conversion in seawater and the consumption of DMSP by these organisms accounts for 1-15% of their carbon demand and most of their demand for sulfur. [6][7][8] The degradation of DMSP by marine bacteria occurs through two competing pathways, with either cleavage or demethylation as the first step. † These authors contributed equally to this work.…”